Analysis system

ABSTRACT

The invention provides an analysis system for capturing target molecules in a sample. The system comprises: (a) supports with a largest dimension of 500 μm or less, wherein each support includes at least one capture analyte bound thereto, said at least one analyte being at least one capture agent exhibiting an affinity for one or more of proteins, antibodies, antibody fragments, DNA aptamers, nucleic acids, small molecules and any other molecules used to bind target molecules; and (b) an arrangement for introducing said sample into contact with said at least one analyte of at least one support in a fluid solution, such that binding of at least one target molecule with at least one analyte is indicative of the presence of said at least one target molecule. The system is distinguished in that: (c) each support comprises identification features for enabling the system to identify the support; (d) the system includes an arrangement for detecting binding of said at least one target molecule with said at least one analyte, the system thereby being capable of associating each support with its corresponding target molecule; and (e) the system further including an additional arrangement for recovering and analysing a remainder of said sample whose molecules are not susceptible to capture by said at least one analyte bound to said supports.

FIELD OF THE INVENTION

The present invention relates to an analysis system for capturing andfiltering target molecules in samples to reduce the complexity of thesamples such that both captured and remaining uncaptured molecules maybe characterised. Moreover, the invention further relates to a method ofcapturing and filtering target molecules in samples to reduce thecomplexity of the samples such that both captured and remaininguncaptured molecules may be characterised.

BACKGROUND TO THE INVENTION

During recent years, there has arisen a considerable interest intechniques and associated systems for determining protein and nucleicacid characteristics of numerous types of organisms, for example, yeast,bacteria and mammals as well as cell lines. There is, for example,currently a need for massively parallel high throughput technologies foridentification and characterisation of proteins (proteomics) inbiotechnological, pharmaceutical, diagnostic, veterinary, petroleum,pulp and paper, food and beverage, and chemical industries.

Similarly, there has also recently arisen a considerable interest intechniques and associated systems for high throughput analysis ofcomplex samples, for example, yeast, bacteria, mammals, cell lines, drugtargets and potential therapeutics. Such analysis includes highthroughput profiling of protein or gene expression providing highvolumes of information about cell events. Mechanisms behind disease andthe effects of therapeutics are associated with protein and geneticprofiles; hence, their analysis provides information for the developmentof diagnostic tests and new drugs. However, attempting to simultaneouslyanalyse all cell events is an exceedingly complicated task, thereforesamples must conventionally first be simplified by, for example,fractionation into related subgroups such as mitochondria or nucleicacids.

For example, the field of proteomics, namely the simultaneous analysisof total gene expression at the protein level, has rapidly become one ofthe leading approaches for studying biological systems and understandingthe relationship between various expressed genes and gene products. Asknowledge about the human genome accumulates, there has been a parallelinterest in developing techniques and associated systems forcorresponding proteomes, namely the entire complement of proteinsexpressed by a particular cell, organism or tissue type. Particularinterest has been shown in the development of techniques for determiningthe characteristics of proteomes associated with particular diseasestates or specific sets of environmental conditions.

Currently, tests for detecting protein characteristics in a samplerequire a large number of experimental steps. The steps includepreparation of the sample by lysis, followed by two-dimensional gelelectrophoresis (2D-GE), post-electrophoresis extraction of the proteinsfollowed by mass spectrophotometry, chromatography, microarrays oradditional electrophoresis methods. A core method presently used inproteomics, namely 2D-GE, is a technique capable of resolving thousandsof proteins and peptides from a single complex mixture in a singleexperiment Proteins are first separated according to their isoelectricpoint, namely the pH at which their net charge is zero, and thenorthogonally separated based on apparent mass using an electrophoresisstep. The individual proteins are revealed as isolated spots on the gelby applying standard staining protocols. However, like many conventionalmethodologies, 2D-GE analysis suffers from a number of seriouslimitations that bring into question the utility of this procedure foradaptation to high-throughput capacity. Such serious limitations includethe number of experimental steps required to identify a protein, poorreproducibility, difficulty in resolution, an inability to visualizelow-abundance proteins, and the high degree of technical skill andsophisticated computational analysis to identify protein spots that arepresent on the gel or blot from one extract but not on the other.

Such aforementioned methods typically result in approximately half ofthe proteins in a given cell being characterised. In order tocharacterise remaining proteins, the above methods are repeated again atleast once. Most researchers believe that 2D-GE, in its present formatrepresents the most significant bottleneck to large-scale proteomicsresearch mainly because it is possible to identify only most abundantproteins in a cell lysate from a 2D gel of a total cellular extract; forexample, only 100 to 600 most abundant proteins represents only afraction of the one billion different proteins/isoforms that may exist.Typically, when analysing a 2D gel of a total cellular extract, proteinsrepresenting only about 250 different gene products are analysed. Sinceprotein separation in 2D gels is based on isoelectric point andmolecular weight, any polypeptides with similar properties areunresolved, namely they will be on the same spot on the gel.

In the discovery of new drug targets, analyses must be expanded beyondthe most abundant and best-characterised proteins of cells. The largenumber of these abundant proteins often causes problems during analysis.Differential fractionation of the cells normally splits the cells intocomponents such as nucleus, cytoplasm, and mitochondria groups which areanalysed using other techniques such. as chromatography andimmunoprecipitation prior to applying standard 2D gels. A consecutiveapproach of splitting samples into components and using severaldifferent methods for analysis for the components is however timeconsuming and requires highly skilled technicians to perform associatedexperiments.

A common way to filter proteins and peptides from a cellular extractprior to their analysis on a 2D gel or on a protein array is an affinitycapture assay. This assay involves using known capture molecules such asantibodies to screen a cell lysate; the antibodies bind their respectivetargets and the remaining corresponding supernatant can be analysedusing a 2D gel procedure as described in a published article Li, J. etal. Mol Cell Proteomics 2002 February 1 (2): pp. 157-68.

There are many examples of affinity capture systems described in theprior art. For example, in a published PCT patent application no.PCT/GB01/04182, there is described Oxford Glycosciences Ltd.'smicroarray affinity capture system in which antibodies are bound to afixed array and used to bind known peptide fragments from a lysedsample.

Moreover, in a published PCT patent application no. PCT/US99/12708 fromImmco Diagnostics Ltd., there is described a method for the quantitationof an analyte in a test sample using an affinity assay. The analyte isbound with a first affinity molecule to form a complex. The complex isthen immobilised to a solid matrix and contacted with a labelled secondaffinity molecule to label immobilized complexes containing the analyte.The amount of analyte in the sample is then quantitated from the amountof label immobilized. In a PCT patent application no. PCT/US98/12843,Ciphergen ProteinChip® describes use of arrays which exhibit specificsurface chemistries to affinity-capture minute quantities of proteins.Such technology requires the use of Surface Enhanced LaserDesorption/Ionisation (SELDI) to identify the captured proteins. Somecommon drawbacks of such techniques are induced denaturation ofpeptides, non-specific binding analytes and interaction of adjacentmolecules on the arrays. Similar issues arise when capturing targetmolecules from complex mixtures of nucleic acids and small moleculessuch as chemical compounds that may be potential therapeutics.

A method for performing affinity assays with a retrievable supportcomprising a magnetic bead, which can reversibly bind to targetmolecule, is described in a patent no. EP0265244 by Amoco Ltd. Beadshave been used to develop a quantitative antibody capture test forC-reactive protein as described in a scientific article Tarkkinen, P. etal., Clin Chem 2002 February 48 (2): pp. 269-77. Both the method and thetest employ capture analytes attached to the beads for capture of thetarget analyte.

Isotope-coded affinity tags (ICAT) have also been developed forselective affinity capture of molecules from complex samples asdescribed in a scientific article Turecek, F. J Mass Spectrom 2002January 37 (1): pp. 1-14.

Affinity capture may also be used for purifying complex mixtures ofnucleic acids or small molecules. In a European patent application no.EP0296557A2, there is described a method of removing undesired singlestranded nucleic acids from a complex mixture of single and doublestranded molecules. The capture analyte consists of single strandednucleic acids bound to water insoluble beads.

A published U.S. Pat. No. 5,759,778 is concerned with a method forisolating and recovering target nucleic acid molecules from a libraryusing biotinylated probes comprising a complementary sequence to thetarget sequence. Moreover, in international PCT patent application no.PCT/US97/02852, there is described a binding assay for detecting smallmolecules such as environmental contaminants, drugs of abuse,therapeutic drugs and hormones. The assay involves use of achromatographic strip containing analyte receptors for binding targetanalytes.

The inventors have appreciated limitations of aforementioned methods,techniques and assays and thereby devised an analysis system that iscapable of addressing these limitations.

SUMMARY OF THE INVENTION

A first object of the invention is to provide an improved analysissystem for the analysis of molecules.

A second object of the invention is to provide an analysis system toimprove the efficiency of analysis of molecules in complex samples.

A third object of the invention is to provide an analysis system toimprove the testing throughput of conventional sample analysisapparatus.

According to a first aspect of the invention, there is provided ananalysis system as defined in the accompanying claim 1.

The system is of advantage in that it is capable of addressing at leastone of the aforementioned objects of the invention.

The invention concerns a method for molecule capture from a complexmixture, where uniquely encoded supports have a capture moleculeattached to a main surface thereof. A multiplexed experiment of hundredsof thousands of tests in one is possible since a large number oflabelled supports and attached capture molecules can be present in theassay simultaneously. Use of such capture molecules in combination withsupports allows identification and recovery of the captured molecules.

In a preferred embodiment of the invention, the primary supports are inthe form of microparticles decreasing the amount of reagents used foreach simultaneous testing process.

The present invention preferably incorporates in an analysis system acoded three-dimensional microparticle array for use in reversibleaffinity capture assays. The system comprises coded microparticles towhich affinity capture analytes are attached, wherein the microparticlesare in solution and/or packed into a column; such an arrangement allowsfor large scale multiplexing of the assays. The analytes may include,but are not limited to, antibodies, antigens, proteins, enzymesubstrate, carbohydrates, peptides, affibodies™, nucleic acids, peptidenucleic acids, cell lines, chemical components, oligonucleotides, serumcomponents, small synthesised molecules, drugs or any derivatives orfragments thereof. This invention offers the benefit of separatingcaptured molecules from a complex sample, namely each encodedmicroparticle carries a different captured target, which can potentiallyprevent unwanted interactions between target molecules and maintains themolecules in solution to prevent denaturation. The coded microparticlesenable the reversibly captured molecules to be identified, recovered andcharacterised without complicated analysis methods such as 2D-GE. Byfiltering out the target molecules, the system reduces sample complexityand hence throughput of the analysis. There are a wide range ofapplications for the analysis system, for example comparative analysisof proteins in cell populations and drug target screening assays.

In another preferred embodiment of the invention, the identificationmeans comprises one or more distinguishing geometrical features, such asshape, size, barcode or dotcode, enabling identification of eachsupport. This allows the use of well-established identificationstandards such as for example barcodes which give good signal to noiseratio and decrease the risk of spectral overlap and false positives.

Other preferred embodiments of the invention, comprises the use of radiofrequency identification transponders (RFID) or optical identification,such as fluorescence or colour coding. The use of RFID gives anadvantage of very large numbers of codes can be used and does notrequire visual communication between the measuring means and theidentifiable support. The use of optical coding on the supports allowsfor combinations of wavelengths or colours not possible with standardfluorescent markers, for example FITC labelled, and allows for using lowcost labelled supports.

According to a second aspect of the invention, there is provided amethod as defined in the accompanying claim 12.

In the second aspect of the invention, there is provided an analysissystem for detecting and quantitating molecule characteristics, whichhas detecting means and identifying means arranged to register twodifferent types of signals, the first signal being associated with thedetection and quantification of activated signal emitting labels and thesecond signal being associated with the reading of sequentialidentification of supports. Such plurality of different types of signaldecreases the potential requirement of using advanced and costly imageprocessing equipment.

The method is of advantage in that it is capable of addressing at leastone of the aforementioned objects of the invention.

It will be appreciated that features of the invention can be combined inany combination without departing from the scope of the invention.

DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings wherein:

FIG. 1 is a plan view and a side view of a single support (microcarrier)comprising a sequential identification;

FIG. 2 is a schematic sectional side view of a single support(microcarrier) with analytes attached thereto;

FIG. 3 is a schematic diagram of an analysis system for an assay,

FIG. 4 is a schematic diagram of an analysis system for a capture assaywith a detailed view;

FIG. 5 is a schematic diagram illustrating the elution of capturedmolecules through a column;

FIG. 6 is a diagram of a flow-based analysis system for analysing thesupports of FIG. 4;

FIG. 7 is a schematic diagram illustrating a planar-based reader forinterrogating the analysis system of FIG. 4; and

FIG. 8 is a schematic diagram illustrating an alternative flush readerfor interrogating the analysis system of FIG. 4.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In FIG. 1, there is shown an illustration of a preferred embodiment of asupport for use in an analysis system according to the invention. Thereis shown a single primary support 1, such a support will also bereferred to as a microcarrier, microparticle or “bead” in the followingdescription. The support 1 can be fabricated from virtually anyinsoluble or solid material, for example one or more of polymers,silicates, glasses, fibres, metals or metal alloys. In the preferredembodiment of the invention, the support 1 is fabricated from a metal,such as gold, silver, copper, nickel, zinc or most preferably aluminium.It is also preferable to use one or more polymers, such as polystyrenes,polyacrylates, polyamides, or polycarbonates when fabricating thesupport 1. The support 1 is preferably either partially or totallycoated in one or more of either of the above-mentioned materials.

The support 1 incorporates an identification feature 2 that is alsoreferred to as an identification code or tag in the followingdescription. Examples of the identification feature 2 may be based onone or more of sequential identification, varied shape and size of thesupport 1, transponders (for example Radio Frequency IdentificationChips, RFIDs) attached to the support 1, and fluorescent coding ordifferent colours of the support 1. Preferably, the identificationfeature 2 is a sequential identification that can be in the shape of atleast one (or any combination thereof) of grooves, notches, depressions,protrusions, projections, and most preferably holes. The identificationfeature 2 being part of the support 1 is advantageous in that there isno need to label each support 1 after manufacture. The sequentialidentification 2 is suitably a transmission optical barcode, which ismachine readable, allowing enhanced signal to noise ratio if read intransmission or reflection. An associated sequential identification codeis thereby recorded in the support 1 as a series of holes using codingschemes similar to those found on conventional bar code systems, forexample as employed for labelling merchandise in commercial retailingoutlets. Such a code allows the use of existing reader technology todetermine the identification feature 2 of the support 1 therebydecreasing the initial investment when adopting technology according tothe invention.

In the preferred embodiment, the primary support 1 is of substantiallyplanar form with at least a principal surface 6 as illustrated inFIG. 1. The support 1 has suitably a width 4 to length 3 ratio in arange of circa 1:2 to circa 1:20, although a ratio range of circa 1:5 tocirca 1:15 is especially preferred. Moreover, the support 1 has athickness 5 that is preferably less than circa 3 μm, and more preferablyless than circa 1 μm. When the thickness is less than circa 1 μm, it hasbeen shown to provide sufficient mechanical support strength forrendering the support 1 useable in harsh experimental conditions. Thelargest dimension 3 of the support 1 is circa 500 μm or less, preferablycirca 300 μm or less, more preferably circa 150 μm or less, mostpreferably circa 100 μm or less, yet more preferably circa 50 μm orless, or preferably even circa 10 μm or less in length. A preferredembodiment of the invention concerns the support 1 having a length 3 ofcirca 100 μm, a width 4 of circa 10 μm and a thickness 5 of circa 1 μm;such a support is capable of storing more than 100,000 differentidentification sequence bar codes 2.

Around 2.5 million supports similar to the support 1 may be fabricatedon a single 3-inch diameter semiconductor-type wafer, for example asilicon wafer, using contemporary established manufacturing techniques.Advantageously, the shape of the support 1 is such that it optimises thenumber of supports 1 manufactured per wafer and also substantiallyoptimises the number of identification codes possible on the supports 1.The support 1 utilises the benefits of a cost effective manufacturingtechnique with the possibility to tailor the design and identificationcoding as required. As described in the foregoing, the shape as well asthe size of the supports 1 may be varied as appropriate usingmicrofabrication manufacturing techniques. Non-exhaustive examples ofpossible shapes are, for example, circular, elliptical, elongated,square, rectangular, multi-cornered or even multi-layered supports ofthe same or different materials. It is also, in some applications,preferable to have the supports 1 in the size of nanoparticles with alargest dimension of circa 500 μm or less; examples of suchnanoparticles comprise cylindrical nanobars. However, a lower limit tosize is governed by sufficient sensitivity of the reaction kineticsbeing achieved.

Conventional photolithography and dry etching processes are examples ofsuch manufacturing techniques used to manufacture and pattern a materiallayer to yield separate solid supports 1 with bar-coded identification2.

A fabrication process for manufacturing a plurality of supports similarto the support 1 involves the following steps:

-   -   (1) depositing a soluble release layer onto a planar wafer;    -   (2) depositing a layer of support material onto the release        layer remote from the wafer;    -   (3) defining support features, including the sequential        identification feature 2, in the support material layer by way        of photolithographic processes and etching processes;    -   (4) removing the release layer using an appropriate solvent to        yield the supports released from the planar wafer; and    -   (5) optionally treating the support material to improve its        attachment properties.

FIG. 2 provides an illustration of how capture analytes 7, such asproteins, antibodies, antibody fragments, DNA aptamers, nucleic acids,affibodies™, small molecules and any other molecules used as captureanalytes 7, are attached to a section 6 of the support 1. Many methodsof chemically treating or physically altering the support material maybe used for the optional step (5) to facilitate the attachment of acapture analyte. Alternatively, the treatment of the support materiallayer, step (5), can be optionally omitted. The treatment of thesupports 1 can be performed after the release from the wafer asdescribed above or alternatively prior to the release from the wafers orearlier in the manufacturing process steps. By modifying the surface 6of the supports 1 or the capture analytes 7, the attachment betweencapture analytes 7 and supports 1 is improved.

Aluminium is a preferred material for the support 1 and there are knownmethods of growing porous surfaces through aluminium anodisation toimprove the attachment properties thereof. Likewise, processes forsealing such porous surfaces are also known. The Applicant has exploitedsuch knowledge to develop a relatively simple process for growing anabsorbing surface having accurately controlled porosity and depth. Suchporous surfaces 6 are capable of achieving a mechanical binding to thecapture analyte 7. Using an avidin-biotin system is another approach forimproving chemical binding between the supports 1 and their associatedcapture analytes 7. The supports' 1 surface 6 may also be treated with abinding material such as silane and/or biotin, to further enhanceattachment properties. The supports 1 preferably have silane baked ontotheir surfaces 6. Attaching linking molecules, for example avidin-biotinsandwich system, to the capture analytes 7 further enhances theirchemical molecular attachment properties.

The enhanced attachment is preferably achieved through having covalentbonds between the attachment surface 6 of the support 1 and the captureanalytes 7. The covalent bonds prevent the capture analytes 7 from beingdislodged from the supports 1 and causing disturbing background noiseduring analysis. There is also a potential problem that loose captureanalytes 7 are capable of preventing the identification of reactionsthat have occurred. It is found to be important to wash the activesupports 1 after attaching capture analytes 7 thereto, to remove anyexcess such analytes 7 that could otherwise increase the noise in theexperiment during analysis. Discrimination of tests using the supports 1is thereby enhanced through a better signal-to-noise ratio.

It will be appreciated that the capture analytes 7 are not limited tothose listed above and can comprise a broad range of compounds capableof being uniquely distinguished and identified. For example the captureanalytes 7 may include antibodies, antigens, proteins, enzyme substrate,carbohydrates, peptides, affibodies™, nucleic acids, peptide nucleicacids, cell lines, chemical components, oligonucleotides, serumcomponents, small synthesised molecules, drugs or any derivatives orfragments thereof. All capture analytes 7 in this broad range may beattached to supports fabricated by steps (1) to (5) above either beforeor after executing photolithographic operations or releasing thesupports 1 from the planar substrate.

Appropriate identification of supports 1 as mentioned above concerns theimportance of using a specific identification for a specific captureanalyte 7. Such an arrangement also allows the use of predeterminedidentification codes 2 for certain capture analytes 7 but also allowsfor matching of identification codes 2 and capture analytes 7 as desiredwhen designing an experiment.

FIG. 3 shows a general method 8 whereby:

-   -   (1) a sample containing target molecules 9 is put in contact        with the capture analytes 7 a bound to the supports 1; and    -   (2) signal emitting labels 10 are bound to capture analytes 7 b.

Each support 1 with its corresponding specific sequential identificationcode 2 has associated therewith a unique capture agent capture analyte 7a, for example a peptide or antibody associated therewith, which bindsto and/or interacts with a specific target molecule 9. The signalemitting labels 10 are for example fluorescent labels. Only supports 1with capture analytes 7 a that have bound to the target molecule 9detected will bind the signal emitting labels 10 and thereby fluorescefrom their emitting labels 11. The result of the test is measured by thedegree of fluorescence of different types of supports 1 with associatedbound molecules. The fluorescent intensity of the bound signal emittinglabels 11 quantifies the level of detected target molecules 9.Experiments where a binary yes/no reaction indication is preferred onlyrequire determination whether or not the supports 1 in the method 8 aresufficiently fluorescent relative to a predetermined fluorescence level.

Alternatively, a test sample containing target molecules is attached toa solid support such as a microtitre plate or tube. A mixture ofsupports 1 with bound capture analytes 7 is added. Each support 1 withits corresponding specific sequential identification features 2 hasassociated therewith a unique capture analyte 7, for example a peptideor antibody associated therewith, which binds to and/or interacts with aspecific target molecule 9. The capture analytes 7 bind to theirrespective target molecules and the unbound supports 1 are washed away.The bound supports 1 are dissociated from the test sample and read bycounting the number of each support 1 type with its correspondingspecific sequential identification features 2 which is proportional orinversely proportional to the amount of target molecules in the testsample. In such a method, signal emitting labels 10 are not used.

In FIG. 4, there is shown a schematic diagram of a first step of anaffinity capture assay. In this example, a panel of 3 different captureanalytes 12, 13, 14 have been bound to supports 1 with 3 differentsequential identification 2 codes. The capture analytes 12, 13, 14 boundto the supports 1 are suspended in liquid and packed into a column 15made of plastic or glass. The sample 16 containing the target molecules17, 18, 19 is introduced to the top of the column 20 and moves throughthe column. The target molecules 17, 18, 19 are captured by theirrespective affinity capture analytes 12, 13, 14, while molecules 21 inthe sample 16 for which there is no capture analyte 7 present will passthrough the column and be collected as an eluent 22 that can besubjected to further analysis.

In FIG. 5, there is illustrated shown a second next step performed inthe affinity capture assay. An elution buffer 23 is added to the top ofthe column 20. This elutes 24 the capture analytes 12, 13, 14 with theirbound target molecules 17, 18, 19 from the column for further analysis.Furthermore, as the capture assay may be reversible, the targetmolecules 17, 18, 19 could be removed from the capture analytes 12, 13,14 for further analysis such as quantitation. The sequentialidentification 2 codes on the supports 1 allow for identification andrecovery of specific target molecules.

The number of different types of supports 1 used for the affinitycapture assay of FIGS. 4 and 5 is dependant on the test throughputrequired, but could be hundreds, thousands or even millions of analytes.The number of the same types of supports 1 employed is dependent,amongst other things, on the quality of statistical analysis desired andthe ease of analysis desired. Signal emitting labels 10 are also addedto the affinity capture assay. These signal emitting labels 10 are usedto indicate interaction, namely bonding, between the capture analytes 7on the supports 1 and the target molecules 9 sought in the analysedsample 16. There are many different ways of adding the signal emittinglabels 10 to the affinity capture assay. They can, for example, be addedto the column 15 separately, be attached to the target molecule 9 to beanalysed prior to the sample 16 being added to the column 15, or beattached to the capture analyte 7 before or after their attachment tothe supports 1. There are also many different ways for the signalemitting labels 10 to indicate that interaction between the captureanalytes 7 and the target molecule 9 in the analysed sample 16. One suchway is for a signal, such as fluorescence or light of other wavelength(colour), to be activated by the signal emitting label 10 if there isinteraction between capture analyte 7, a matching target molecule 9 andthe signal emitting label 10. Alternatively the signal emitting labels10 are activated before any interaction with the target molecule 9. Whenthere is an interaction between the capture analyte 7 and the targetmolecule 9, the active signal emitting label 10 is released from theother molecules deactivating its signal. This would result in adetection that is opposite to the ones discussed previously, namely theabsence of a signal indicates that a reaction has occurred on a supportin, for example, a yes/no experiment. Similarly, a decrease in thefluorescent signal from the emitting labels 11 can be an indicator ofthe amount of target molecule 9 present in the analysed sample 16introduced into the column 15.

Applications for the affinity capture assay include protein profiling ofa cell, tissue, organ or whole organism or a cellular extract, lysate orprotein fraction derived therefrom. Such an assay can also be used fordetermining the epitope profile of cells, tissues, organs and wholeorganisms and cellular extracts, lysates or protein fractions derivedtherefrom Such applications are relevant for analysis of drug targets,libraries of potential therapeutic agents and for diagnostics. Since thesystem of the invention reduces the complexity of samples by firstfiltering out known target molecules 9 with their respective captureanalytes 7, it therefore enriches the sample for low quantity moleculeswhose identify and function may then be more easily elucidated.

By way of example, a sample, for instance derived from a cell culture,is first lysed to release all the proteins and peptides in solution,namely >10,000 proteins per cell. The lysate is introduced to the columnshown in FIG. 4, on which there are supports 1 containing bound captureanalytes 7. The capture analytes 7 were previously selected to capture,for example, specific peptide isoforms. The resulting sample eluent thencontains only those molecules not captured by the capture analyte 7,namely the sample is enriched for uncharacterised molecules and theexperiment can now focus on characterising the unknowns. A furtheradvantage is that by reducing the total number of input molecules to theexperiment, researcher using the system of the invention are less likelyto detect those molecules that would overlap in analysis, for examplepeptides which electrophorese to the same spot on 2D-GE

The large number of supports 1 with sequential identification codes 2available means that as new target molecules 9 are identified andcapture analytes 7 developed for them, they can be added to the affinitycapture assay, therefore providing a means for enriching the samples forlow abundance molecules.

Reader systems for use with the reversible affinity capture assaysupports will now be described. The Applicant has developed two classesof reader systems. These are based on flow cells for handling thesupports 1, and on planar imaging of plated-out supports 1.

A flow-based reader system, similar in construction to a flow cytometer,can be used to draw through thousands of supports 1 per second, therebyreading simultaneously the sequential identification code 2 of eachsupport 1 and the results of its associated test result. The test resultis measured as a yes/no binary result or by the degree of fluorescence11.

In FIG. 6, the flow-based reader system is shown indicated generally by25. At a downstream end, the reader system 25 comprises a measuring unitindicated by 26 for reading supports 1 conveyed in operation in fluidflow from an injecting nozzle 27 at an upstream end to the measuringapparatus 26 at the downstream end. The apparatus 26 includes a readingzone 28, a reader unit 29, a light source 30, a detector unit 31, asignal emitting unit 32 and a processing unit 33.

A sample 38, for example a liquid comprising a plurality of the supports1 dispersed therein, is introduced into the focussing zone 34. Moreover,a flow of carrier fluid 35 is generated along a tube 36 in a directionfrom the upstream end towards the downstream end. Preferably, thecarrier fluid 35 flowing in operation along the tube 36 is a liquid.Alternatively, the fluid 35 can be a gas at reduced pressure relative tothe nozzle 27 so that liquid bearing the supports 1 to an exit aperture37 is vaporised at the aperture 37, thereby assisting to launch supports1 into the tube 36. Whereas it is easier to establish a laminar flowregime within the tube 36 when fluid flowing therethrough is a liquid,gas flow through the tube 36 potentially offers extremely fast support 1throughput and associated interrogation in the reading zone 28.

The reader 25 is designed to induce the supports 1 to flow along acentral region of a tube 36 through the defined interrogation zone 28.By utilizing an accelerated sheath fluid 35 configuration and theinjecting nozzle 27, the supports 1 injected into the central region ofthe tube 36 are subjected to a hydrodynamic focusing effect 39 causingall the supports 1 to align lengthwise, namely axially, and to passthrough a well-defined focal point 40 in the interrogation zone 28downstream from the exit aperture 37. The distance between the exitaperture 37 and the interrogation zone 28 must be sufficiently long todissipate any turbulence caused by the injection nozzle 27. Thissufficient length allows for a substantially laminar flow of the readingfluid 35 and hence provides the supports 1 with a non-oscillatingmovement past the focal point 40. If required, the nozzle 27 can beprovided with a radially symmetrical arrangement of feed tubelets fromthe focussing zone 34 so as to obtain a more symmetrical velocityprofile within the tube 36. In an interface surface region in closeproximity to the peripheral surfaces of the tube 36, fluid velocityprogressively reduces to substantially zero at the interior surface ofthe tube 36.

The supports 1 are ejected from the exit aperture 37 and are swept inthe flow 35 along the tube 36 into the reading zone 28 and eventuallytherepast. When one or more of the supports 1 enter the reading zone 28,light from the source 30 illuminates the one or more supports 1 at thefocal point 40 so that they appear in silhouette view at the reader unit29. The reader unit 29 generates a corresponding silhouette signal thatis communicated to the processing unit 33 for subsequent imageprocessing to determine the sequential identification 2 of the supports1. Preferably, the light source 30 emits light in a plane A-A that issubstantially perpendicular to the samples' flow 35 direction and fromtwo different radial directions, the radial directions preferably havinga mutual angle separation, for example with a mutual angular separationof circa 45° separation. Such an arrangement of support 1 illuminationin the focal point 40 enables the supports 1 to be identifiedirrespectively of their rotational position along their longitudinalaxis.

For each support 1 transported through the zone 34, the processing unit33 is programmed to determine the sequential identification 2 of thesupport 1 with its corresponding magnitude of fluorescence. The readerunit 29, located substantially at an opposite side of the interrogationzone 28 relative to the light source 30, reads the light that passesthrough one or more supports 1 at the focal point 40. The detector unit32 detects any fluorescence occurring in the zone 28 and generates acorresponding fluorescence signal that is subsequently received by theprocessing unit 33. The detector unit 31 measures the magnitude of theintensity of the fluorescent signal 11 that is given off by theactivated signal labels 10 on the supports 1. This intensity indicatesthe degree of reaction that can be extrapolated to determine therelative amount of reactive target molecule 9 present in the sample.Moreover, the processing unit 33 is also connected to an associateddatabase relating the sequential identification 2 with a test providedby its associated capture analytes 7.

Examples of successful experiments of sorting supports I withfluorescent signal indicating successful capture of wanted molecule hasbeen performed on the Union Biometrica, COPAS™ flow cytometer at a rateof 20 supports 1 per second and sorting one support 1 into a well of amicrotitre plate with 96-well format. These results in the COPAS™ can bequalitative or quantitative depending on the experiment requirements.Other flow cytometers also successfully used for measuring and sortingout positive response supports 1 are the MoFlo™ from DakoCytomation andthe FACScan™ from Becton Dickenson.

A feature in the form of a marking at one end of each support 1 is usedto indicate to the reader unit 29 how to interpret the read information.This allows the support 1 to be read from either direction along itslongitudinal axis. The marking is also susceptible to being used toincrease the number of possible sequential identification codes on asupport 1 to be greatly in excess of 100,000. For example, employingfour different markings on separate sets of supports 1 is capable ofincreasing the number of identification combinations of supports toabout 400,000. An alternative feature to indicate how information codesare to be read is to start each block with 0's and end the blocks with1's, or vice versa. Further alternatives of these features arepreferably error correction data, for parity bit checks and/or forwarderror correction, thereby improving testing reliability.

As an alternative to the flow-based reader system of FIG. 6, a planarreader system can be employed, wherein:

-   -   (a) the supports 1 are plated out onto a planar substrate; and        then    -   (b) fluorescence microscopy and associated image processing are        employed to read the bar codes of the supports and the results        of their associated tests.

In FIG. 7, there is shown a planar reader system indicated generally by41. After the capture assay has been completed as described withreference to FIG. 6, the supports 1 are deposited on the planarsubstrate 42. Preferably, the planar substrate 42 is fabricated from apolymer, glass or silicon-based material, for example a microscopeslide. A planar measuring unit 43 arranged to perform conventionalfluorescence microscopy is used to analyse the support-plated substrate42 systematically, measuring the level of fluorescence of emittinglabels 11 thereon and also the sequential identification 2 of thedifferent supports 1 of the support-loaded substrate 42. Normally, allthe supports 1 on the loaded substrate 42 are analysed to verify thetotal quality of the experiment. In cases where it is desirable to savetime and/or to increase processing capacity, software executing in aprocessing unit 44 of the reader system 41 can preferably be configuredto analyse only the supports 1 whose emitting labels 11 fluoresce, forexample by virtue of their fluorescent signal labels 10, indicating thatan interaction with the target molecule 9 has occurred. The analysis ofthe loaded substrate 42 using the planar measuring unit 43 is a verycost effective, easy to perform and suitable way to multiply theanalysing capacity for low to medium sample numbers in the range of, forexample, single figures to a few thousand supports on each substrate 42.

The planar measuring unit's 43 reader unit 45 for image-processing isused to capture digital images of each field of the substrate 42 towhich supports 1 have become affixed. Digital images thereby obtainedcorrespond to light transmitted through the substrate 42 and itsassociated base plate 46 and then through the supports 1 rendering thesupports 1 in silhouette view; such silhouette images of the supports 1are analysed by the reader unit 45 in combination with a processing unit44. The sequential identification 2 of the supports 1 may also be readby reflected light. The sequential identification 2, for example abar-code, associated with each support 1 is hence identified from itstransmitted or reflected light profile by the reader unit 45. The signalemitting unit 32 generates a fluorescent signal, which signal makes thesignal emitting labels 11 on supports 1 that have interacted with thetarget molecules 9 fluoresce. A detector unit 31 detects the magnitudeof fluorescence from activated supports 1 to identify the degree ofreaction. The fluorescent signal integrated over activated supports' 1surface 6 is recorded in association with the identification bar-code 2to construct data sets susceptible to statistical analysis.

The processing unit 44 is connected to the light source 30, the signalunit 32, the reader unit 45, and the detector unit 31 and to a display47. Moreover, the processing unit 44 comprises a control system forcontrolling the light source 30 and the signal unit 32. The lightsilhouette or reflected light and fluorescent signals from the supports1 pass via an optical assembly 48, for example an assembly comprisingone or more lenses and/or one or more mirrors or electrochemicalshutters and filter wheels, towards the detector unit 31 and reader unit45. By way of example, a mirror assembly as shown can be employed in thereader system 41. A mirror 49 is used to divide the optical signals intotwo paths and optical filters 50, 51 for filtering out unwanted opticalsignals based on their wavelength. Alternatively, the light source 30and signal unit 32 can be turned on and off at intervals, for examplemutually alternately. Signals are received from the reader unit 45 anddetector unit 31, these signals being processed and correspondingstatistical analysis results presented on a display 47. Similar numbersof each type of supports 1 are required to give optimal statisticalanalysis of experiments. Such statistical analysis is well known in theart.

In FIG. 8, there is shown the flush cell reader system and indicatedgenerally by 100. The flush system 100 is configured in a similar mannerto the planar system 41 but employs a flushing action to introducesupports 1 to be read. After the aforementioned capture assay has beencompleted, the supports 1 are flushed into the reader cell 110 via asample inflow tube 120. Preferably, the reader cell 42 is fabricatedfrom a clear polymer, glass or silicon-based materials, for examplePerspex. The measuring unit 43 is arranged to perform conventionalfluorescence microscopy and is used in operation to analyse the supports1 that have settled onto a base of the reader cell 110, therebymeasuring the level of fluorescence of supports 1 thereon, and also thecorresponding sequential identification 2 of the supports 1 thereon.Normally, all the supports 1 on the loaded reader cell 110 are analysedto verify the total quality of the experiment. In cases where therecould be an interest in saving time and/or increasing processingcapacity, the software of the processing unit 44 is preferablyconfigurable to analyse only the supports 1 that emit a fluorescentsignal, namely their including fluorescent signal labels 10 fluoresce,indicating that an interaction with the target molecule 9 has occurred.The analysis of the loaded reader cell 110 using the planar measuringunit 43 is a very cost effective, easy to perform and suitable way tomultiply the analysing capacity for low to medium sample numbers in therange of, for example, single figures to a few thousand supports on eachreader cell 110.

The planar measuring unit's 43 reader unit 45 for image-processing isused to capture digital images of each field of the reader cell 110 towhich supports 1 have settled. Digital images thereby obtainedcorrespond to light transmitted through the reader cell 110 and its baseplate 46 and then through the supports 1 rendering the supports 1 insilhouette view; such silhouette images of the supports 1 are analysedby the reader unit 45 in combination with a processing unit 44. Thesequential identification 2 of the supports 1 may also be read byreflected light. The sequential identification 2, for example abar-code, associated with each support 1 is hence identified from itstransmitted or reflected light profile by the reader unit 45. The signalemitting unit 32 generates a fluorescent signal, which signal makes thesignal emitting labels 11 on supports 1 that have interacted with thetarget molecules 9 fluorescence. A detector unit 31 detects themagnitude of fluorescence 11 from activated supports 1 to identify thedegree of reaction. The fluorescent signal integrated over activatedsupports' 1 surface 6 is recorded in association with the identificationbar code 2 to construct data sets susceptible to statistical analysis.

The processing unit 44 is connected to the light source 30, the signalunit 32, the reader unit 45, and the detector unit 31 and to a display47. Moreover, the processing unit 44 comprises a control system forcontrolling the light source 30 and the signal unit 32. The lightsilhouette or reflected light and fluorescent signals from the supports1 pass via an optical assembly 48, for example an assembly comprisingone or more lenses and/or one or more mirrors or electrochemicalshutters and filter wheels, towards the detector unit 31 and reader unit45.

By way of example, a mirror assembly is shown. A mirror 49 is used todivide the optical signals into two paths and optical filters 50, 51 areused to filter out unwanted optical signals based on their wavelength.Alternatively, the light source 30 and signal unit 32 can be turned onand off at intervals, for example mutually alternately. Signals arereceived from the reader unit 45 and detector unit 31, which areprocessed and corresponding statistical analysis results presented on adisplay 47. Similar numbers of each type of supports 1 are required togive optimal statistical analysis of experiments. Such statisticalanalysis is well known in the art.

Once the supports 1 have been identified by the system, buffer isflushed through the reader cell 110 via a buffer inflow tube 130 and theread supports 1 are washed from the reader cell 110 via an outlet tube140. The next sample of supports to be read are then introduced via thesample inflow tube 120.

It will be appreciated that modifications can be made to embodiments ofthe invention described in the foregoing without departing from thescope of the invention as defined by the appended claims.

1. An analysis system for capturing target molecules in a sample, thesystem comprising: (a) supports with a largest dimension of 500 μm orless, wherein each support includes at least one capture analyte boundthereto, said at least one analyte being at least one capture agentexhibiting an affinity for one or more of proteins, antibodies, antibodyfragments, DNA aptamers, nucleic acids, small molecules and any othermolecules used to bind target molecules; (b) engaging means forintroducing said sample into contact with said at least one analyte ofat least one support in a fluid solution, such that binding of at leastone target molecule with at least one analyte is indicative of thepresence of said at least one target molecule; characterised in that:(c) each support comprises identifying means for enabling the system toidentify the support; (d) the system includes interrogating means fordetecting binding of said at least one target molecule with said atleast one analyte, the system thereby being capable of associating eachsupport with its corresponding target molecule; and (e) the systemfurther including analysis means for recovering and analysing aremainder of said sample whose molecules are not susceptible to captureby said at least one analyte bound to said supports.
 2. A systemaccording to claim 1, wherein at least one target molecule captured ontoits corresponding at least one analyte is reversibly bound thereto suchthat said at least one reversibly bound molecule is susceptible to beingrecovered, characterised and quantitated using said interrogating means.3. A system according to claim 1, wherein the amount of target moleculepresent in the sample is quantitable from the amount thereof bound tosaid at least one capture analyte.
 4. A system according to claim 1,wherein the analysis means for analysing the remainder of the sampleincludes one or more of the following for performing such analysis:microarrays, mass spectrophotometry, 2D-GE, chromatography, sequencing,flow cytometry and immunoprecipitation.
 5. A system according to claim1, wherein the largest dimension of the support is less than 300 μm. 6.A system according to claim 1, wherein the largest dimension of thesupport is less than 150 μm.
 7. A system according to claim 1, whereinthe largest dimension of the support is less than 50 μm.
 8. A systemaccording to claim 1, wherein the identifying means comprises one ormore of distinguishing geometrical features, such as shape, size,barcode or dotcode, enabling identification of each support.
 9. A systemaccording to claim 1, wherein at least one of the identification meansis a radio frequency identification transponder (RFID).
 10. A systemaccording to claim 1, wherein at least one of the identification meansis an optical identification, such as fluorescence or colour based. 11.A system according to claim 1, wherein the fluid solution is a liquid.12. A method of capturing and filtering target molecules in a sample,the method including the steps of: (a) providing supports with a largestdimension of 500 μm or less, wherein each support includes at least onecapture analyte bound thereto, said at least one analyte being at leastone capture agent exhibiting an affinity for one or more of proteins,antibodies, antibody fragments, DNA aptamers, nucleic acids, smallmolecules and any other molecules used to bind target molecules; (b)introducing said sample into contact with said at least one analyte ofat least one support in a fluid solution, such that binding of at leastone target molecule with at least one analyte is indicative of thepresence of said at least one target molecule; characterised in that themethod further comprises the step of: (c) providing each support withidentifying means for enabling identification of the support; (d)detecting binding of said at least one target molecule with said atleast one analyte, thereby associating each support with itscorresponding target molecule; and (e) recovering and analysing aremainder of said sample whose molecules are not susceptible to captureby said at least one analyte bound to said supports.
 13. A methodaccording to claim 12, wherein at least one target molecule capturedonto its corresponding at least one analyte is reversibly bound theretosuch that said at least one reversibly bound molecule is susceptible tobeing recovered, characterised and quantitated using interrogatingmeans.
 14. A method according to claim 12, wherein the amount of targetmolecule present in the sample is quantitable from the amount thereofbound to said at least one capture analyte.
 15. A method according toclaim 12, wherein in step (e) the remainder of the sample is analysedusing one or more of the following: microarrays, mass spectrophoto,etry,2D-GE, chromatography, sequencing, flow cytometry andimmunoprecipitation.
 16. A method according to claim 12, wherein thelargest dimension of the support is less than 300 μm.
 17. A methodaccording to claim 12, wherein the largest dimension of the support isless than 150 μm.
 18. A method according to claim 12, wherein thelargest dimension of the support is less than 50 μm.
 19. A methodaccording to claim 12, wherein the identifying means comprises one ormore of distinguishing geometrical features, such as shape, size,barcode or dotcode, enabling identification of each support.
 20. Amethod according to claim 12, wherein at least one of the identifyingmeans is a radio frequency identification transponder (RFID).
 21. Amethod according to claim 12, wherein at least one of the identifyingmeans is an optical identification, such as fluorescence or colourbased.
 22. A method according to claim 12, wherein the fluid solution isa liquid.
 23. (canceled)
 24. (canceled)